Elemental flow cytometer

ABSTRACT

An elemental flow cytometer includes a device to vaporize, atomize, and ionize material and an introduction system for introducing packets of discrete entities into said device to vaporize, atomize and ionize materials to vaporize, atomize and ionize the entities in the packets. A spectrometer is adapted to individually analyze elemental composition of one or more of the vaporized, atomized and excited or ionized packets.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent Ser. No. 13/351,909,filed Jan. 17, 2012, which is a continuation of U.S. patent applicationSer. No. 12/332,812, filed Dec. 11, 2008, now abandoned, which is adivisional of U.S. Pat. No. 7,479,630, issued Jan. 20, 2009, which is anon-provisional of U.S. Patent Application Ser. No. 60/555,952, filedMar. 25, 2004, all of which are hereby incorporated by reference.

The entire contents of U.S. patent application Ser. No. 09/905,907,filed Jul. 17, 2001 and entitled Elemental Analysis of TaggedBiologically Active Materials (published as US 2002/0086441); and Ser.No. 10/614,115, filed Jul. 3, 2003 and entitled Elemental Analysis ofTagged Biologically Active Materials (published as US 2004/0072250) arehereby incorporated by reference.

The entire contents of U.S. Pat. No. 6,524,793, filed Jun. 18, 1999 andentitled Multiplexed Analysis of Clinical Specimens Apparatus andMethod; International Patent Application Publication WO 98/33203,published Jul. 30, 1998, and entitled Gate for Eliminating ChargedParticles in Time of Flight Spectrometers; and each of the publicationscited in the Reference Section herein are hereby incorporated byreference.

FIELD OF THE INVENTION

The invention features apparatus and methods for sequentially analyzingparticles, for example single cells or single beads, by spectrometry. Inparticular, the invention provides elemental-flow cytometers.

BACKGROUND OF THE INVENTION

The ability to analyze single particles, for example single cells orsingle beads, is a useful tool in the health sciences, in human andanimal food sciences, in environmental sciences, forensic sciences, andin genomics and proteomics.

In the health sciences, cells are recognized as members of certainclasses, for example normal cells or cancerous cells for diagnostic orbiomedical research purposes. Cells carry multiple antigens orbiomarkers [1], either extracellularly or intracellularly [2], which canbe quantified or qualified for clinical medicine [3] or biomedicalresearch [4] purposes. These methods are useful for development ofpharmaceutical products particularly in the development of cell basedassays and toxicity studies.

For example, chronic lymphocytic leukemia (CCL) is recognized as aunique disorder of B-cells [5, 6]. CCL is a disease with an uncertainclinical picture, and is often misdiagnosed resulting in inadequatetreatment. However, a more detailed study of a patient's cellularimmunophenotypic profile allows reclassification of the patient, whichleads to a more personalized diagnosis and treatment. Suchclassification requires multi-targeted analysis of many markers on acell membrane as well as in-cell antigens, their qualitative andquantitative description, and consideration of minute concentrationvariances.

Other examples in the health sciences include the analysis of singlecells in the subclassification of non-Hodgkin's lymphoma. In addition,single cell analysis is useful in immunophenotyping of helper T-cells,and the determination of the ratio of CD4 to CD8 T-cells, for indicationof the HIV progression in HIV positive patients. Further, the techniquecan be used to analyze single cells from patients with renal, cardiacand bone marrow transplants, for discriminating between graft rejectionsand viral infections in post-operative patients.

In human and animal food sciences, the analysis of single cells can beused to detect artificial hormones, pesticides, herbicides orantibiotics. Finally, in environmental sciences, the analysis of singlecells can detect toxic waste, for example, in plant or bacterial cells.

A known method of analyzing single cells is by a fluorescence activatedcell sorter (FACS). FACS is a technology to measure biologicalproperties of cells by scanning single cells as they pass through alaser beam. Cells are usually stained with one or more fluorescent dyesspecific to cell components of interest, for example, receptors on thecell surface and DNA of the cell nucleus, and the fluorescence of eachcell is measured as it traverses the excitation beam. Since the amountof fluorescence emitted is proportional to the amount of fluorescentprobe bound to the cell antigen, antibodies conjugated to fluorochromesare routinely used as reagents to measure the antigen both qualitativelyand quantitatively on and in the cell. Primarily, researchers use thesorting function of the FACS machines to investigate cell receptors andother membrane antigens on a specific cell population. It can be usedfor antibody screening in multiple cell lines simultaneously (forexample, a transfected cell line expressing the antigen of interest anda control cell line not expressing the antigen). In its simplified flowcytometry function, FACS machines are used mostly without sorting, whichallows for example the use of fixed permeabilized cells and analysis ofintracellular antigens. Many routine flow cytometry methods thatidentify antigens expressed on the cell surface and within the cellusing specific antibodies, as well as general immunoassay methods forclinical diagnostics and treatment have been developed. Some of theminvolve multiplexing through the use of different fluorochromes andlasers. Deficiencies of this approach are related to limitations anddifficulties of cell staining methods and spectral overlap offluorochromes. Other measurable optical parameters include lightabsorption and light scattering, the latter being applicable to themeasurement of cell size, shape, density, granularity, and stain uptake.

U.S. patent application Ser. No. 09/905,907, published under US2002/0086441 on Jul. 4, 2002, and Ser. No. 10/614,115, describe labelingof analytes for analysis by mass spectrometry. Biologically activematerials (for example, antibodies and aptamers) are labeled andconjugated to analytes prior to analysis.

SUMMARY OF THE INVENTION

One aspect of the invention is an analytical instrument. The instrumenthas a sample introduction system for generating a stream of particlesfrom a sample and an ionization system for receiving particles in thestream. The ionization system is operable to atomize particles in thestream as the particles are received from the sample introduction systemand to ionize atoms from the atomized particles. The instrument has anion pretreatment system and a magnetic sector mass analyzer comprisingan array detector. The ion pretreatment system is adapted to transportions generated by the ionization system to the magnetic sector massanalyzer. The magnetic sector mass analyzer is adapted to detect atransient signal of at least one element from individual particles fromsaid stream by performing mass analysis on the ions from the atomizedparticles. The magnetic sector mass analyzer is adapted determine anamount of said at least one element from an individual particle usingthe transient signal detected during mass analysis of the ions from saidindividual particle.

In another broad aspect, the invention provides an apparatus forintroducing particles sequentially and analyzing the particles (forexample, single particles such as single cells or single beads), byspectrometry. The apparatus, an elemental flow cytometer, is aninstrument comprising: a means for introducing single particlessequentially, a means to vaporize, atomize, and excite or ionize theparticles or an elemental tag associated with an analyte on theparticles, and a means to analyze the elemental composition of thevaporized, atomized, ionized and/or excited particles, or an elementaltag associated with the particles.

It is to be understood that although the term “means for introducingsingle particles sequentially” is used, this may encompass introductionof a predetermined number of particles (for example, 2 or more) indiscrete ‘packets’.

It is also to be understood that the term “means to vaporize, atomize,and excite or ionize” includes means where atomization may not benecessary, so that the term may or may not encompass vaporizationfollowed by ionization directly. In some applications, such as forexample optical emission spectrometry (OES), it is not essential toionize the sample; emission from atomic species can be sufficient. ForOES, it is necessary only to excite the atoms (or ions) to causeemission. Thus, for example, “vaporize, atomize and ionize” should beunderstood to mean vaporize, atomize and ionize (for mass spectrometry)or excite (either or both atoms and ions) for OES.

Another aspect of the invention is an analytical instrument. Theinstrument has a sample introduction system for generating a stream ofparticles from a sample. An ionization system receives particles in thestream. The ionization system is operable to atomize particles in thestream as the particles are received from the sample introduction systemand to ionize atoms from the atomized particles. The instrument has anion pretreatment system and a mass analyzer. The ion pretreatment systemis adapted to transport ions generated by the ionization system to themass analyzer. The mass analyzer is adapted measure the amount of atleast one element in individual particles from the stream by performingmass analysis on the ions from the atomized particles.

Another aspect of the invention is an instrument for performingmulti-parametric quantitative analysis of particles in a stream ofparticles. The instrument has a sample introduction system forgenerating a stream of particles from a sample. A particle analyzer isadapted to measure the amount of each of a plurality of at least fivedifferent tags in each of a plurality of particles in the stream ofparticles produced by the sample introduction system. The particleanalyzer has a detector adapted to generate signals corresponding toeach tag. The signals generated by the detector corresponding to each ofthe tags is independent from the signal generated by the detectorcorresponding to the others of the tags.

In another broad aspect, the invention provides a method for analyzingparticles that have been introduced sequentially, such as single cellsor single beads, by spectrometry. A trigger will report the ion cloudarrival with following analysis, including for example initiation ofdata acquisition. Triggering may be based, for example on lightscattering or on an ion current change or ion composition change.

Another aspect of the invention is an elemental flow cytometer,comprising: a means for introducing particles sequentially into a deviceto vaporize, atomize and excite or ionize the particles, or an elementaltag associated with the particles; a device to vaporize, atomize andexcite or ionize the particles, or an elemental tag associated with theparticles, downstream of the means for introducing particlessequentially; and a spectrometer to analyze the vaporized, atomized andionized and/or excited particles, or the elemental tag associated withthe particles.

Another aspect of the invention is a mass-spectrometer-based flowcytometer, comprising: a means for introducing particles sequentiallyinto a device to vaporize, atomize and ionize the particles, or anelemental tag associated with the particles; a device to vaporize,atomize and ionize the particles, or an elemental tag associated withthe particles, downstream of the means for introducing particles,sequentially; and a mass spectrometer operatively connected anddownstream of the device to vaporize, atomize and ionize.

Another aspect of the invention is a mass-spectrometer-based flowcytometer, comprising: a means for introducing particles sequentiallyinto a device to vaporize, atomize and ionize the particles, or anelemental tag associated with the particles; a device to vaporize,atomize and ionize the particles, or an elemental tag associated withthe particles, downstream of the means for introducing particlessequentially; an ion pretreatment device operatively connected anddownstream of the device to vaporize, atomize and ionize; and a massspectrometer operatively connected and downstream of the ionpretreatment device. The ion pretreatment device may be provided as apart of the mass spectrometer, preferably upstream of the mass analyzersection thereof.

Another aspect of the invention, is an optical emissionspectrometer-based flow cytometer, comprising: a means for introducingparticles sequentially into a device to vaporize, atomize and excite orionize the particles, or an elemental tag associated with the particles;a device to vaporize, atomize and excite or ionize the particles, or anelemental tag associated with the particles downstream of the means forintroducing particles sequentially, and an optical emission spectrometerto analyze the vaporized, atomized and excited or ionized particles, orthe elemental tag associated with the particles downstream of the deviceto vaporize, atomize and excite or ionize the particles.

Another aspect of the invention, is a method of analyzing particles thathave been introduced sequentially into a device to vaporize, atomize andexcite or ionize, comprising: sequentially introducing particles orparticles associated with an elemental tag, into a device to vaporize,atomize and excite or ionize the particles or the elemental tagassociated with the particles; and introducing the vaporized, atomizedand excited or ionized particles, or the elemental tag associated withthe particles into a spectrometer.

The labeling or tagging of the single particles with elemental tags canbe done, for example, using the methods and system disclosed in U.S.Ser. No. 09/905,907 and U.S. Ser. No. 10/614,115, both applications ofwhich are herein incorporated by reference. U.S. Ser. No. 09/905,907 andU.S. Ser. No. 10/614,115 describe methods and systems for the analysisof biologically active materials conjugated to analytes by massspectrometry. Other methods of labeling or tagging the particles willalso serve. If, for example, the particles are beads, the particlesthemselves can be labeled either on the surface or within their bodies,as disclosed herein.

Another aspect of the present invention is to provide kits havingreagents for carrying out the methods of the present invention andinstructions for these methods.

Another aspect of the present invention is to provide beads with anaffinity substance as a carrier to measure an analyte in a sample,further comprising an elemental label or tag. The elemental tag can beon the analyte, on the affinity substance or (and) on or in the beaditself.

Yet another aspect of the invention is an elemental flow cytometer. Theelemental flow cytometer includes a device to vaporize, atomize, andionize material. and an introduction system for introducing packets ofdiscrete entities into said device to vaporize, atomize and ionizematerials to vaporize, atomize and ionize the entities in the packets. Aspectrometer is adapted to individually analyze elemental composition ofone or more of the vaporized, atomized and excited or ionized packets.

Other objects and features will in part be apparent and in part bepointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a flow cytometer according to theinvention.

FIG. 2 is a schematic diagram of an embodiment of amass-spectrometer-based flow cytometer according to the invention.

FIG. 3 is a schematic diagram of an embodiment of a optical emissionspectrometer (OES)-based flow cytometer of the invention.

FIG. 4 is a schematic diagram of a single-particle injector according tothe invention.

FIG. 5 is a calibration curve for flag-BAP using agarose beadimmobilization with α-BAP primary and Au-tagged α-mouse secondaryantibodies.

FIG. 6 is a plot of Fluorokine bead assay, detecting TNF-.alpha. andIL-6 simultaneously using distinguishable (Eu and Tb) elemental tags onthe corresponding primary antibodies.

FIG. 7 is a plot of an ELISA based assay coupled to ICP MS showing thesimultaneous quantitation of two proteins.

FIG. 8 shows a schematic diagram of a sample introduction system

FIG. 9 shows overlaid results of measuring ion signals as a function oftime for direct injection of a standard solution of 100 ppt Rh (1% HNO₃)and of a MOTE cell suspension for which the surface antigen CD33 wastagged with a Au particle.

FIG. 10A shows oscilloscope output of an Ar₂ ⁺ signal and FIG. 10B showsoscilloscope output of an Au⁺ from MO7e cell introduction for which thesurface antigen CD33 was tagged with a Au particle.

FIG. 11A shows an analog signal from an oscilloscope registered whilecontinuously monitoring Na⁺ produced by a cell suspension in 30 mM CaCl₂and FIG. 11B shows a corresponding signal produced just by the buffer 30mM CaCl₂.

FIG. 12 shows comparative data for analysis of cell surface proteins andintracellular proteins by both conventional FACS and by the method ofthe present invention.

FIG. 13A shows Si⁺ signal and FIG. 13B shows Tb⁺ signal for stobersilica particles grown in the presence of a Tb solution.

DEFINITIONS

ICP-MS: is an Inductively-Coupled Plasma Mass Spectrometer.

FACS: is a Fluorescence Activated Cell Sorter.

Single particle: is a single entity (item, material, element, substance,structure, organism, matter, body, article or thing) or a single packetof a predetermined number of discrete entities (for example, 2, 3 or 4),and includes but is not limited to single cells, single beads, singlebacteria, single viral particles, single pollen particles, singlemicroscopic insects such as dust mites.

Tag (or label): a chemical moiety that provides a distinguishable signalof the presence of the analyte or analyte complex with which it isassociated, as for example through linkage to an affinity product thatin turn recognizes the analyte or analyte complex. As disclosed herein,the tag (which is also called an “elemental tag”) can contain an elementor an isotope (or multiple copies thereof) that provide thedistinguishable signal. A tag can include for example an element orisotope of an element that is associated with an analyte or analytecomplex and which is measured to determine the presence of the analyte.A tag can also include, for example, any distinguishable component(e.g., an element or isotope or multiple copies thereof) that isprovided on the surface or within the body of, or is otherwiseassociated with, a particle and serves to distinguish that particle fromother particles.

TOF-MS: is a Time-of-Flight Mass Spectrometer

DESCRIPTION OF SPECIFIC EMBODIMENTS INCLUDING THE BEST MODE CURRENTLYCONTEMPLATED BY THE INVENTORS

The elemental flow cytometer of the present invention provides for theidentification and quantitative analysis of particles that have beenintroduced sequentially into a device to vaporize, atomize and excite orionize them, for example individual cells or microscopic beads, bymeasuring the elemental composition of the single particle (or anydistinctive part of cell or bead), or a tag or label associated with ananalyte located on or in the cell or bead by employing themass-to-charge ratio or optical emission of the disintegrated tagelements. The tag can be of any chemical nature, as it is only itselemental composition that is important. In comparison, the chemicalstructure of the appropriate tag is absolutely critical to provide aunique fluorescence in FACS.

The elemental flow cytometer includes:

means for introducing particles sequentially (for example,

cell-by-cell or bead-by-bead), preferably adapted for discrete eventanalysis;

means to vaporize, atomize and excite or ionize the particles,

or an elemental tag (or classifiable elemental composition) associatedwith an analyte of interest on or in the particles to quantify theanalyte of interest associated with the particles;

and means for registering the information on elemental composition ofthe particles, or an elemental tag associated with an analyte on theparticles. This can be done, for example, by mass spectrometry (MS) orby optical emission spectrometry (OES).

Elemental flow cytometers according to the invention are quantitativeanalytical instruments [7]. They can perform the task of quantitative orqualitative analysis of biological or environmental samples usinganalytical methods [8].

Beads with an affinity substance can be used as carriers to measure ananalyte in a sample. The placement of the elemental tag or label can beon the analyte, on the affinity substrate, and/or on or in the beaditself Specific embodiments of the elemental flow cytometer include: (1)a mass spectrometer based flow cytometer (MS FC) and (2) an opticalemission spectrometer based flow cytometer (OES FC).

A mass spectrometer based flow cytometer (MS FC) comprises:

means for introducing particles sequentially:

means to vaporize, atomize and ionize the particles and/or any tags thatmay be associated with the particles; and

a mass spectrometer to analyze the elemental composition of thevaporized, atomized and ionized particles, and/or any tags that may beassociated with the particles.

MS FCs according to the invention can further comprise ion pretreatmentdevices, for pretreatment of ions prior to analysis by the massspectrometer.

The means to vaporize, atomize and ionize the single particles mayinclude glow discharge, graphite furnace, and capacitively coupledplasma devices, or other suitable devices. Preferably, the means tovaporize, atomize and ionize the single particle includes an inductivelycoupled plasma (ICP) device because it has a capacity to disintegrate,vaporize, atomize and ionize cells and beads during their shortresidence time in the plasma and because the ICP is particularlytolerant of concomitant materials, is robust to changes of thecomposition of the plasma gases, and is a highly efficient atomizer andionizer.

The ion pretreatment device acts, inter alia, as an interface betweenatmospheric conditions in the vaporizer/atomizer/ionizer and the vacuumin the mass spectrometer. In addition, the very strong ion currentoriginating from this source is dominated by space charge, which couldbe reduced by an accelerating potential and/or by rejection of majorplasma ions on the basis of their mass-to-charge ratio (Ar⁺, forexample). In the case of a TOF MS, the ion pretreatment device alsoconditions the ion flow for the needs of the TOF mass analyzer. Forexample, it will narrow the ion energy distribution and focus theparallel ion beam close to the axis of the mass analyzer.

The mass spectrometer can be any mass spectrometer. For example, it canbe a quadrupole, magnetic sector with array detector, 3D Ion Trap orLinear Ion Trap mass spectrometer. Preferably it is a time of flightmass spectrometer (TOF MS). TOF MS is a simultaneous analyzer. It isable to register all masses of interest in one particle simultaneously.

The optical emission spectrometer based flow cytometer (OES FC)comprises:

a means for introducing particles sequentially; a means to vaporize,atomize and excite or ionize the particles, and/or any tags that may beassociated with the particles; and

an optical emission spectrometer to analyze the elemental composition ofthe vaporized/atomized and excited or ionized particles and/or any tagsthat may be associated with the particles.

The means to vaporize, atomize and excite or ionize the single particlesmay include glow discharge, graphite furnace, and capacitively coupledplasma devices, or other suitable devices. Preferably, the means toatomize and ionize the single particles includes an inductively coupledplasma (ICP) device because it has a capacity to disintegrate, atomizeand excite or ionize cells and beads during their short residence timein the plasma and because the ICP is particularly tolerant ofconcomitant materials, is robust to changes of the composition of theplasma gases, and is a highly efficient atomizer and ionizer.

Processes implemented by elemental flow cytometers according to theinvention can also include an in-line lysis step between the means forsingle particle introduction and the means to vaporize, atomize andionize.

The embodiments will now be described in detail.

In a most general aspect, the present invention provides an elementalanalyzer as a detector for a flow cytometer. FIG. 1 shows schematicallya cytometer 100 suitable for use implementing methods of analysisaccording to the invention. Cytometer 100 comprises means 102 forintroducing particles sequentially, for example a cell or particleinjector 171 (FIGS. 2, 3, 4), operatively connected upstream of a device104 for vaporizing, atomizing and exciting or ionizing particles orelemental tags associated with the particles. The elemental compositionof the particle or elemental tag is determined by a spectrometer 106operatively connected to the device 104. Spectrometer 106 may, forexample, include an optical spectrometer 157, which detects the emissionfrom excited atoms and/or ions, or a mass spectrometer 116 which detectsthe ions.

In one embodiment the present invention provides a mass-spectrometerbased flow cytometer (MS FC) 101. A schematic representation of such anembodiment is given in FIG. 2.

Referring to FIG. 2, mass-spectrometer based embodiment 101 of cytometer100 comprises means 102 for introducing particles sequentially, forexample a cell or particle injector 171, operatively connected upstreamof device 104 for vaporizing, atomizing and exciting or ionizingparticles or elemental tags associated with the particles, namely aninductively coupled plasma (ICP) vaporizer/atomizer/ionizer. In theembodiment shown, means 102 comprises optional in-line lysis system 110.

Ion pretreatment device 112, in this instance comprising vacuuminterface 114, high-pass filter 116 and gas-filled “cooler” cell 118, isoperatively connected downstream of the ICP vaporizer/ionizer.

Time-of-flight (TOF) mass spectrometer 106, 161, 126 is operativelyconnected downstream of the ion pretreatment device. Use of massspectrometer-based cytometer 101 according to such embodiments toanalyze single particles can provide greatly improved accuracy, largedynamic range and high sensitivity, compared to prior art systems. Inaddition, because a large number of distinguishable elements andisotopes can be used as tags, and because the mass spectrometer provideshigh abundance sensitivity (exceedingly small overlap of signal onadjacent mass/charge detection channels), it facilitates a higher orderof multiplexing (simultaneous determination of multiple analytes, eachdistinguishably tagged) than prior art fluorescence-based detection flowcytometers. Further, because of the high resolution of adjacentmass/charge detection channels and the large linear dynamic range of themass spectrometer, the instrument provides for a large dynamic rangeboth for a given analyte and between analytes. Thus, in many instancesgeneric tagging moieties can be used in analyses in which the copy-countof the analytes differs dramatically; this distinguishes the method fromconventional fluorescence detection methods for which the composition ofthe several fluorophores used for multiplex assay must often be adjustedfor a particular assay to provide emission intensities of similarmagnitude to minimize spectral overlap. Thus such embodiments canprovide researchers and clinicians substantially improved analytical andprognostic capabilities.

Another important application of cytometers according to this embodimentof the invention is to multiplex assay distinguishable beads, where thebeads are distinguished by their elemental compositions and haveattached affinity products that recognize an antigen in the sample intowhich they are introduced, where the antigen is then further recognizedusing a sandwich (or other) assay employing yet a furtherdistinguishable element.

Significant components of the mass spectrometer-based flow cytometer 101of FIG. 2 and methods of use will now be described in detail.

Tagging

In certain cases the particle (for example a single cell) will notrequire Tagging

In some cases a particles will not require tagging. For example, if asingle cell contains or is bound to an element that is detectableagainst the background by mass spectrometry, no tagging is required. Forexample, for the analyses of bacterial or plant cells that accumulateelemental species in bioremediation, additional tagging would not berequired. Further, the intracellular accumulation of metal, for exampleplatinum- or gold-containing drugs would not require additional tagging.

In Cases where Single Particles Require Tagging

Tagging of particles can be done by many methods as is known to those ofskill in the art. For example, fluorescent dyes which have asuccinimidyl ester moiety react efficiently with primary amines ofproteins (antibodies) to form stable dye-protein conjugates. In a firststep to tag DNA, an amine-modified nucleotide, 5(3-aminoallyl)-dUTP, canbe incorporated into DNA using conventional enzymatic tagging methods.In a second step, the amine-modified DNA can be chemically tagged usingan amine-reactive fluorescent dye. Biotinilation of antibodies can becarried out using sulfhydryl-directed solid-phase chemistry. Thesemethods are well established and are available in kit formats fromdifferent companies, including for example Molecular Probes Inc, PierceChemical Company, and others. Specific chemical reactions are known inradioimmunochemistry. For example, radionuclides (88/90)Y and (177)Lucan be used to tag antibodies using cyclic diethylenetriaminepentaaceticacid anhydride (cDTPA), isothiocyanatobenzyl-DTPA (SCN-Bz-DTPA), or1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA)(PMID: 14960657).

Elemental analysis of tagged biologically active materials has beendisclosed in the incorporated references, U.S. patent application Ser.Nos. 09/905,907 and 10/614,115. Tagged biologically active materials,for example, antibodies and aptamers, etc., that react specifically withcellular components can be used to tag cells. Other affinity productsare known to those skilled in the art. For example, they may includeantigens, RNA, DNA, lipoproteins, glycoproteins, peptides, polypeptides,hormones, etc.

Although in many applications of systems and methods according to theinvention it is convenient to tag each biologically active material (forexample an antibody, aptamer or antigen) with a single element orisotope, it should be readily appreciated by those skilled in the artthat an antibody or antigen may be tagged with more than one element. Asthere are more than 80 naturally-occurring elements having more than 250stable isotopes, there are numerous elements, isotopes, and combinationsthereof to choose from. Within limits prescribed by the need to havedistinguishable tags when in combination, this will allow forsimultaneous detection of numerous biologically-tagged complexes. It isadvantageous if the relative abundance of the tag elements issufficiently different from the relative abundance of elements in agiven sample under analysis. By “sufficiently different” it is meantthat under the methods of the present invention it is possible to detectthe target antibody or antigen over the background elements contained ina sample under analysis. Indeed, the difference in inter-elementalratios of the tagged antibody or antigen, and the sample matrix can beused advantageously to analyze the sample.

It is feasible to select elemental tags, which do not produceinterfering signals during analysis. Therefore, two or more analyticaldeterminations can be performed simultaneously in one sample. Moreover,because the elemental tag can be made containing many atoms, themeasured signal can be greatly amplified.

The use of multiple copies of the element or isotope per tag can improvethe sensitivity linearly, particularly, for example in the employment ofICP-MS embodiments of the invention. For multiplex assay of up to 23simultaneous analytes, the tags can be conveniently constructed usingthe natural isotopic distributions of, for example, Ru, Rh, Pd, Ag, In,La, Ce, Pr, Nd, Sm, Eu, Th, Dy, Ho, Er, Tm, Yb, Lu, Hf, Re, Ir, Pt andAu. These elements, which are expected in most instances to be uncommonin biological samples, each have at least one isotope with naturalabundance greater than 10% that is not significantly interfered by theothers or by the oxide or hydroxide ions of the others. For thoseisotopes of lower natural abundance (e.g., ¹⁴³Nd 12.2%), tagging withthe isotopically enriched isotope provides an obvious sensitivityadvantage. Where a higher order of multiplexing is desired, the use ofcommercially-available enriched isotopes (of which there may be as manyas 167 of 55 elements that are not expected to be common in biologicalsystems) offers a possibility (depending, of course, on availability,cost and isotopic purity). For example, there are as mentioned at least35 isotopes of the lanthanides and noble metals alone that may beobtained in enriched form, are not expected to be common in biologicalsystems and are largely independent with respect to mutual interference(though some care in the selection of the tagging protocol is to betaken where large differences in copy counts of the analytes occur; forexample, if the copy count of an analyte tagged with .¹⁶⁹Tm is 1000times greater than for an analyte tagged with .¹⁸⁵Er, .¹⁶⁹TmO⁺ willinterfere significantly with the determination of .¹⁸⁵Er⁺ since TmO⁺ istypically about 0.07% of the Tm⁺ signal (though, as for FACS, some ofthis interference can be corrected mathematically since the fractionalformation of oxide ions is stable and can be calibrated). In specialcircumstances, it might be feasible to tag a given biologically activematerial with more than one element or isotope (for example, there arein theory 20 distinguishable 3-isotope tags that can be constructed from4 isotopes).

The invention allows the development of a novel powerful technique tomeasure biological properties of cells by analyzing single cells as theypass through an ICP. When using antibodies as the affinity product(biologically active material) the amount of a tag element detected bythe mass spectrometer is proportional to the amount of tagged affinityproduct bound to the cell. Antibodies conjugated to the elemental tagare routinely used as reagents to measure the antigen both qualitativelyand quantitatively, for example acquiring the patient's immunophenotypicprofile, which is almost unlimited in the number of markers of interest.Another advantage offered by the invention is a reduced need to enhancethe antibody signal by “sandwich” immunostaining” which can result inanalytical errors.

Methods according to the invention are distinct from the approach ofconventional methods (such as fluorescence, radioimmunoassay,chemiluminescent assay) that are challenged by overlap of detectorsignals, limited dynamic range, time-sensitive signals, and in someinstances sensitivity. Accordingly, the method offers the potential formassively multiplexed assay (limited principally by the independence andcross-reactivity of the affinity chemistry) with essentially no signaloverlap. Where the elemental (isotopic) tags are quantitativelyassociated with specific affinity products, the quantitativecharacteristic of ICP-MS offers a novel opportunity for absolutedetermination of multiple antigens simultaneously.

The method and apparatus can, for example, detect as few as 100 copiesof each tag per cell. It is estimated that for the detection of as fewas 100 copies of each tag per cell, at least 70 atoms per tag will berequired.

The invention provides the feasibility to perform massively multiplexedbead assays. Current fluorescence-based flow cytometers are frequentlyused for bead assays. In this application, beads are typically labeledwith 2 fluorochromes in varying ratios, typically providing up to about100 distinguishable beads as determined by the fluorophore emissionratio (see, for example, the incorporated reference, U.S. Pat. No.6,524,793 and references therein). Each bead also has attached affinityproducts (e.g., antibodies) that recognize an analyte in a solution inwhich the bead is placed, each bead of different “colour” having anaffinity product for a different analyte. Once exposed to the samplesolution, the captured analyte is then sandwiched with another antibodyhaving a third fluorophore reporter. Thus, in the flow cytometricanalysis, the beads can be mixed, the copy-count of the analyte captureddetermined by the emission of the third fluorophore and the identity ofthe analyte determined by the ratio of the emissions of thebead-labeling fluorophores. Accordingly, the conventional fluorescencedetector flow cytometer can perform multiplexed bead assays to as high100 order (the number of distinguishable “colours”, though in practicemuch fewer are used (presumably because of signal overlap, which limitsthe measurement accuracy (and thus confidence in the identification ofthe bead) when the ratio of the fluorescence emission intensity is large(e.g., one or two orders of magnitude, depending on the emissionwavelength distributions).

A similar method can be implemented using mass spectrometer-based flowcytometers according to the invention, with the advantage that thedegree of multiplexing can be vastly increased and the overlap ofsignals can be virtually eliminated from concern. For example, the beadcan incorporate (either on its surface or, probably more conveniently,within its body, mixtures of elements or isotopes that can be used toreport the identity of the bead. Assuming that the detector has adynamic range of 3 orders of magnitude and that factors of 3 in relativesignal can be reliably determined, 2 elements incorporated into the beadallows 63 distinguishable beads. Under the same assumptions but using 5element labels provides 32,767 distinguishable beads, and if the dynamicrange is 5 orders of magnitude, the same 5-element labels provide for248,831 distinguishable beads. Furthermore, these few labeling elementscan be selected so that signal overlap is nonexistent (e.g., by choosingthem such that they appear at mass differences greater than a few atomicmass units), which enables the large dynamic range of detection. Thesandwich assay for the analyte captured by the bead employs a yetdifferent element tag, which also is readily distinguished from thebead-labeling elements. Further, in this configuration each bead cancontain several affinity products to attach several different analytesper bead, each recognized by a sandwich assay using a yet differentelement, providing for multiplex assay both between beads and on asingle bead. One anticipated application is for a 96-well plate (or384-well, or 1536-well) for which a differently-labeled bead is providedto each well, and multiplexed element-tagged immunoassay on the beadsurface in each well is conducted. The entire contents of the plate (96,or 384 or 1536 wells) can then be pooled and the result analyzed by flowcytometry, thus providing a type of mass spectrometer “plate reader”(where the bead identity, as determined by its elemental composition,identifies the well in which the assay took place).

Means for Introducing Particles Sequentially

The sample introduction system 102 can comprise several devices that arecurrently in use with other flow cytometry sample introduction systems.For example, there currently exist several cell or particle injector 171systems in use for flow cytometry, including various formats of sheathflow injection. Because of considerations for solvent loading of the ICP(typically optimum for 25 to 80 μL/min), the “flow in air” (or in theinstance of the ICP, “flow in argon”) injector 171 may in somecircumstances be considered most appropriate (though some improvementover current designs may be preferred, in order to minimize cellagglomeration). All sample introduction devices suitable for thepurposes disclosed herein; including ICP devices, will serve, regardlessof whether they now exist or are hereafter developed or improved.

For the feasibility experiments that we report below, a small volumespray chamber (similar in concept to a design reported by J. L Todoliand J. M Mermet, J. Anal. At. Spectrometry 2002, V17, 345-351) wasemployed, having a drain to remove condensed liquid (of which there wasessentially none at the suspension flow rates used) and having no gasoutlet except into the ICP.

It is noted that, compared to the FACS method for which carefulalignment of the particles with the excitation laser is important, thepresent method allows relaxation of the alignment of the particles withthe vaporizer, atomizer and ionizer (unless light scattering is used asthe particle detection trigger; see later). This is because, especiallyfor the ICP instance, the precise position of the particle within theinjector tube feeding the ICP is of little importance to the detectedsignal (in part because the central channel flow containing the particleexpands dramatically upon heating and in part because virtually all ofthe central channel flow is inhaled into the sampler of the ICP-MSvacuum interface, though only the predominantly central portion issubsequently transmitted through the skimmer; in any event, thereappears to be substantial mixing of the central channel flow prior tosampling into the vacuum interface).

It is desirable that the entire particle introduced to the ICP bevaporized, and at least partially atomized and ionized, so as to enabledetermination of the element tags contained within the particle(intracellular tags, or bead labels). Current wisdom holds that solidparticles (e.g., of glasses or geological materials) smaller than about1 μm diameter, and liquid aerosols smaller than about 10 μm diameter,are efficiently vaporized, atomized and ionized in the ICP, while largerparticles may be only partially volatilized. This is presumably due tothe short transit time of the particle through the ICP, for which theheat transfer to a large particle is insufficient to allow completevaporization, atomization and ionization. Thus, it is propitious to usebeads having a diameter smaller than about 1 μm diameter (for example,we used stober silica particles of about 150 nm diameter in ourfeasibility studies described below). However, cells are frequentlylarger than 10 μm diameter. Nonetheless, our feasibility experiments,described below, suggest that cells larger than this perceived minimumare, in fact, efficiently vaporized, atomized and ionized, from which weinfer that, upon the rapid heating during transit through the ICP, thecell explodes into fragments that are small enough to be vaporized,atomized and ionized. It remains possible that in certain instances theparticles may be too large to allow efficient vaporization, atomizationand ionization, which could be indicated by the failure to observe anintracellular tag or the element labels of a bead. In this instance,several ion source parameters (gas flow, power, sampling depth) can beadjusted to alleviate this deficiency. Alternatively, an in-line lysiscomponent can be employed.

In-Line Lysis

In-line lysis system 110 may be advantageously employed in somecircumstances. For example, in the event that whole cell introduction isnot viable, use of an in-line lysis system can be advantageous. This maybe done by any method suitable for the purposes disclosed herein,including a number of methods now known to persons skilled in the art,including acidification of the sheath flow fluid to cause cell collapseor high purity (low conductivity) water sheath flow to induce rupture ofthe cell by osmotic pressure. In this instance, the elemental tags willbe retained and transmitted to the device to vaporize, atomize andionize the sample, though the transient pulse may be broadened slightlyby diffusion in the flow stream.

Means for Vaporizing/Atomizing/Ionizing

Any means 104 suitable for the purposes disclosed herein can be employedto vaporize, atomize and excite or ionize the particle or the elementaltag associated with the particle; for example, graphite furnace, glowdischarge and capacitively coupled plasma. Preferably, thevaporizer/atomizer/ionizer is an inductively coupled plasma. In someinstances, vaporization, atomization and ionization and/or excitationcan occur in different devices and at different times (e.g., within agraphite furnace for vaporization in combination with ICP foratomization and ionization and/or excitation.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a preferredmeans of determining the elemental composition, especially ultra-tracecomponents, of materials. It has found acceptance in variousapplications including environmental (e.g., drinking, river, sea andwaste water analyses), geological (e.g., trace element patterning),clinical (e.g., determination of trace metals in blood, serum and urine)and high purity materials (e.g., semiconductor reagents and components)analysis.

ICP-MS couples an inductively coupled plasma ionization source to a massspectrometer. Briefly, a sample, most commonly an aerosol produced bynebulization, is injected into a high temperature atmospheric pressureplasma obtained by the coupling of radio frequency (rf) energy into theflowing argon gas stream. The resultant plasma is characterized by ahigh temperature (ca. 5000K) and relatively high concentration (ca. 10¹⁵cm.⁻³) of equal numbers of electrons and positive ions. Provided thatthe particles of the nebulized sample are small enough, as describedabove, the sample is promptly vaporized, atomized and ionized as itflows through the plasma. The efficiency of ionization is inversely andexponentially dependent on the ionization potential of the elements,with the majority of the periodic table being nearly 100% ionized. Theresultant plasma containing the ionized sample components is extractedinto vacuum where the ions are separated from neutral species andsubjected to mass analysis. The “mass fingerprint” identifies theelements contained in the sample. The detected signal is directly andquantitatively proportional to the concentration of the elementalcomposition of the sample. The particular attributes of the method ofnote include: wide linear dynamic range (9 orders of magnitude),exceptional sensitivity (sub-part per trillion, or attomole/microlitre,detection), high abundance sensitivity (<10⁻⁶ overlap between adjacentisotopes for quadrupole analyzers), counting-statistics-limitedprecision, absolute quantification, and tolerance of concomitant matrix.

ICP-OES is another preferred method of performing the analyses describedabove; it is of particular merit when the solids content of the sampleis greater than about 1% (for homogeneous liquid introduction rate ofthe order of 1 mL/minute). The conditions employed in the ICP arecomparable to those described for the ICP-MS method. Detection of theemission from excited neutral atoms and ions in the ICP provides for thequantitative determination of the elemental composition of the sample.Most current ICP-OES instruments provide array detection for truesimultaneous determination across most of the periodic table. In manyfavorable instances, ICP-OES retains some of the desirablecharacteristics of ICP-MS, including wide dynamic range andwell-resolved detection channels. In other instances, there is potentialfor inter-element or molecular emission interference, though in suchinstances alternate emission wavelengths are frequently available. Theprincipal deficiencies for the application considered here are itsgenerally lower sensitivity (in some instances limited by backgroundemission signals) and its inability to distinguish isotopes of a givenelement. Nonetheless, ICP-OES is perceived to be more simple to use,more robust, and less expensive than ICP-MS, and hence may haveapplication for the present method.

Ion Pretreatment Device

In some circumstances, as for example in MS FC, an ion-pretreatmentdevice 112 may be used to condition the ions for the mass analyzer.Because the mass spectrometer operates at reduced pressure (typicallyless than 10⁻⁴ torr) and the ion sources noted above typically operateat higher pressure (e.g., atmospheric pressure for the conventionalICP), one function of the ion-pretreatment device is to efficientlytransport the ions derived from the sample through a pressure reductionstep (the vacuum interface). It is desirable in this step, andsubsequently, to increase the ratio of ions to neutrals that aresubsequently transmitted to the mass analyzer. Ion optical components(ion lenses) typically serve this function, by localizing the ions andallowing the neutrals to be removed through vacuum pumps. An additionalfunction of the ion optics is to condition the ion beam (in space andenergy) to match the acceptance of the mass analyzer.

High-pass filter 116 and ‘cooler’ cells 118 are only two of the manysuitable forms of pretreatment that now exist; doubtless other formswill hereafter be developed. Any devices or methods suitable for thepurposes herein will serve.

Due to the short residence time of a single particle passing through theplasma, two separate ion handling (pretreatment) and mass separatingtechniques may be used.

A gain of two orders of magnitude relative to current ICP-TOF-MSinstruments, which means about one order of magnitude greater thancurrent quadrupole systems is also desired. The mass spectrometer-basedflow cytometer is ideal for the detection of heavy atom tags. It issufficient to determine only the mass range above ca. 100 amu. One ofthe most significant impediments to improved sensitivity is space chargerepulsion of the dominant Ar⁺ ions (m/z=40). Since the method is notlimited by the conventional elemental analysis demands (the mass rangeof the typical elemental analyzer is from m/z=4 to m/z=250), it ispossible to optimize the ion optics for the transmission of high massions.

While a conventional ICP-MS having simultaneous detection capability(for example, an ICP-TOF-MS 126 or ICP-ion trap-MS) is as a detector ofthe MS FC 101, it should be realized that the requirements of the MS FC101 are quite distinct from those of the conventional elemental analysisapplication. In particular, in the MS FC application the elements to bedetermined (as tags or labels) can be selected with advantage to bethose above, say, 90 atomic mass units (amu, dalton, Thomson). In suchinstance, there is no need to provide simultaneously optimum sensitivityfor low mass (e.g., Li, B, Na, Mg, Co, etc.) and high mass (e.g., thelanthanides and noble metals).

One approach employing a TOF analyzer 126 is to accelerate the ion beamrelatively early in the plasma expansion because an accelerated beam hasa higher space-charge-limited ion current and to high-pass filter thebeam. This can be through the use of a quadrupole-type device, which isnot pressurized. The depleted ion beam can then be decelerated (evencollisionally cooled in a pressurized multipole, which could potentiallyalso provide ion-bunching) prior to injection into the TOF. It isanticipated that the space-charge limit of such a continuous extractionbeam is sufficiently high to allow a ten-fold improvement in sensitivityfor the higher mass ions.

An alternate, or concomitant approach, is to pulse-extract the ion beam.Since lower mass ions are accelerated to high velocity in a givenextraction field, the Ar⁺ ions (and lower mass ions, which can bediscarded) run ahead of the higher mass ions of interest. Preferably,the front-running ions can be discarded using an orthogonal pulse,similar to “SmartGate” of the GBC TOF, (see, e.g., WO 98/33203) but inthe ion optics region. The transmission window does not need to beprecisely defined in this instance, as it is sufficient to interceptions<100 amu. A downstream cooling cell could still be used to bunch theions and normalize their energies. If the orthogonal pulser isproblematic, the entire pulse-extracted ion beam can be run into the TOFextraction region, with the deficiency that more-narrow mass windowswill be simultaneously injected into the TOF. Simple calculations (whichoverestimate the potential by at least some margin) indicate that a 15%duty cycle pulse-extractor could yield up to 28-fold (m/z=100) and12-fold (m/z=238) sensitivity improvement over current (80 Mcps/ppm)quad systems. This assumes 100% transmission efficiency through the ionoptics and 100% duty cycle of the TOF (requiring bunching).

The ion pretreatment device may also include a particle event trigger,which triggers instrument mass selection and detection systems toacquire data from discrete particles, and keeps the instrument idlebetween events. As is know to those skilled in the art, this can be donein many different ways.

Therefore, the ion pretreatment device may comprise:

a vacuum interface;

a high-pass mass filter downstream of the vacuum interface; and

a gas filled ion cooler cell downstream of the vacuum interface.

Among the distinctions that simplify the design of the MS FC 101according to the invention relative to a conventional elemental analyzerare the relative invariance of the sample (cells or beads in a knownbuffer) that simplify the need for an ionizer design (e.g., ICP) that istolerant of various sample types and matrices, the relative (withrespect to the total ion current of the ICP) invariance of the totalelemental composition of the sample that relieves the need to providecompensation for inter-element matrix suppression effects (recognizingthat, for example, Na and Ca will be significant components of cells),and to a large extent (depending on the selection of tag elements) theneed to compensate for the presence of spectral interferences due toargides, oxides and doubly charged ions. Thus, MS FCs according to theinvention can be advantageously adapted to suit the cytometricapplication but not for the general elemental analytical applicationbecause of the selectability of the elements to be determined. Forexample, conventional elemental analysis by ICP-MS is compromised by themutual repulsion of ions following extraction into the vacuum system;this space charge effect, well known to those skilled in the art,derives principally from the overwhelmingly large flux of lower massions that derive from the plasma support gas or the sample solvent suchas O⁺, Ar⁺, ArO⁺, Ar₂ ⁺, and in some instances lower mass ions thatderive from other sample matrix components such as Na⁺, Ca⁺, Cl⁺. Itwill be recognized that the most significant of these ions that form thebulk of the space charge effect are low mass ions, being below about 80amu. Thus, advantage is to be had by eliminating such low mass ions asearly as possible following extraction into the vacuum system becausedoing so will alleviate the space charge and its associated effectivepotential field barrier that suppresses transmission of other ions.Several schemes for achieving this relief can be conceived, includingthe use of a high-pass mass filter such as a quadrupole device that isoperated to transmit ions above, say 80 amu. Notably, a quadrupole canbe operated at the pressures extant in the ion optics region (typicallyabout 10⁻³ torr). An additional advantage of such an ion pretreatmentdevice for the present application is that it can also be operated tosimultaneously provide a low pass mass filter function (that is, abandpass between a selected low mass and a selected high mass). In theinstance that a time-of-flight mass analyzer is used, this bandpass canprovide an improvement in duty cycle (resulting in improved sensitivity)because it minimizes the incursion of the arrival of high mass ions froma previous pulse into the arrival time distribution of the current pulseand also the incursion of low mass ions from the previous pulse into thearrival time distribution of the current pulse (where “pulse” means thepacket of ions that are injected into the flight tube of the time offlight mass analyzer). Further, acceleration of the ions as soon aspossible upon their entrance to the vacuum system (or near the pointwhere the debye length of the plasma is comparable to the dimensions ofthe apparatus or lenses) can further mitigate the space charge effects.However, in the instance that the ions are subsequently decelerated (forexample, in the acceleration region for the TOF), the space chargeeffects can return and reassert themselves resulting in reduction ofsensitivity and, in the instance of the TOF, reduced mass resolution dueto energy broadening in the direction of the flight tube. Hence, thehigh pass mass filter, which can be functional at relatively high ionkinetic energy if appropriately designed, can be operated in concertwith acceleration optics to mitigate space charge effects bothimmediately downstream of the vacuum interface and further downstream,for example in the acceleration region of a TOF mass analyzer 126.

It is further advantageous, as is well known to those skilled in theart, that reduction of the axial ion energy by collisions with anon-reactive buffer gas in a pressurized multipole cell (a “cooler”cell) 118 provides improved resolution and sensitivity for TOF massanalysis (also expected to be true for an array-detector magnetic sectormass analyzer). Here again, the high pass mass filter 116, which shouldprecede the “cooler” cell 118, can be operated in concert with the“cooler” cell 118 with advantage, since bandpassing the ions prior tothe “cooler” cell 118 will mitigate to large extent space charge effectsthat otherwise would be detrimental (i.e., cause loss of sensitivity) inthe “cooler” cell (which would happen because the ions are slowed bycollisions in the “cooler” cell, and slowing them without first removingthe bulk of the low mass “space charge inducing” ions causes an abruptappearance of a significant defocusing space charge field near theentrance of the “cooler” cell).

As is known to those skilled in the art, in certain instances advantageis also to be had in including reactive gases in the “cooler” cell 118in order to transform ions that are isobaric and thus are interfering orare interfered (reference U.S. Pat. No. 6,140,638). Further, the“cooler” cell can also be operated in a trap-and-pulse mode that couldbe optimized for synchronous operation with a TOF acceleration pulse toprovide improved duty cycle (and hence sensitivity) for that massanalyzer. Thus, the MS FC 106 can incorporate with advantage ionacceleration optics and a high pass mass filter.

For several mass analyzer embodiments, including in particular the TOFand array-detector magnetic sector configuration, the use of agas-filled “cooler” cell is also advantageous. For the TOF configurationin particular, the high pass mass filter could with advantage beoperated as a bandpass mass filter with both a low and a high masstransmission limit. As is known to those skilled in the art, the highpass mass filter and “cooler” cell can be combined as a single unit(cf., U.S. Pat. No. 6,140,638).

Advantage is also to be had, to minimize the volume of data collected toinclude only the most significant data or, in the instance of a massanalyzer (such as TOF) which is constrained by a duty cycle, tocoordinate the measurement of data with the passage of a particle ofinterest through the detector system. In the conventional FACS method,this coordination is accomplished most often by the measurement of lightscattering as the particle passes through the excitation region; thenature of this light scattering (forward and side light scatter) canprovide information on the size and granularity of the particle whichalso has diagnostic value. In the MS FC or OES FC method, lightscattering can be similarly used.

Where the source of excitation is an ICP, the scattering event can bedetected prior to vaporization of the particle; hence a delaycorresponding to the time or spatial delay required for signalgeneration. For OES FC this is the time or distance required forvaporization, atomization, ionization and emission; for MS FC anadditional delay corresponding to the transit time of the extracted ionsfrom the region of ionization to the mass analyzer is required. Thoseskilled in the art will realize that for continuous monitoring massspectrometers, for example an array detector magnetic sector massanalyzer, this delay should be applied to the arrival of the ions at thearray detector. For other mass analyzers, for example TOF and ion traps,the delay is applied to the device that introduces the ions into themass analyzer, for example the acceleration region preceding the flighttube of the TOF or a pulsing lens that introduces ions to an ion trap,to which the subsequent mass analysis and detection is synchronized.

Other methods of providing a trigger for data collection arecontemplated for the MS FC 106. For example, it is expected that thepassage of a particle through the ionizer (for example, the ICP) willcause an abrupt and consequent change in the mass distribution of themajor ions that are extracted (for example, the dominant Ar⁺ signal inICP-MS could be suppressed with concomitant formation of C⁺, H⁺, Na⁺,Ca⁺, etc.). It is thus expected that the ion current ejected or thespatial position of this ion current ejection (due to differences in thestability characteristics of ions of different masses) from, forexample, the high pass mass filter, will change significantly and can bedetected with one or more electrodes within or external to, for example,the high pass mass filter. Further, the magnitude or duration of thecurrent change detected may be correlated with the size or content ofthe particle and could provide further diagnostic information.

Other trigger devices are contemplated, including, for example, adetector that measures changes in the ion current or impedence ormagnetic field associated with the ion beam extracted into the vacuumsystem.

Optionally, various components, including for example a high mass filterand a gas-filled ion cooler, may be provided in a single housing. Thiscan provide, for example, improved durability, as well as improvedoperating, handling and installing qualities.

Mass Spectrometer

The pretreated ion cloud may be analyzed with a simultaneous massanalyzer. Sequential mass analysis (e.g., through the use of quadrupoledevices) is also possible. Examples of simultaneous mass analyzersinclude TOF, 3D trap and Linear Trap.

In some instances where the MS FC 101 method is to be used to bestadvantage (e.g., multiplex assay of individual particles), asimultaneous mass analyzer is preferred. For example, in the instance ofthe use of an ICP as the vaporizer, atomizer and ionizer, the transientsignals from a single particle may last for a period in the range 20 to200 microseconds, which can be insufficient to allow quantitativemultiplex assay using a sequential mass analyzer, for example aquadrupole mass analyzer. In such instances, examples of preferred massanalyzers include TOF, array-detector magnetic sector, 3D ion trap andlinear ion trap. In other instances where the period of the transientsignal is significantly longer, either by the nature of the device tovaporize, atomize and ionize or by broadening of the transient signal,for example through transport of the vaporized particles, atoms or ionsthrough a length of tubing or through collisional processes (such asthose reported by D. R. Bandura, V. I. Baranov and S. D. Tanner in J.Anal. At. Spect. 2000, V15, 021-928), a sequential mass analyzer mayfind utility.

At the current state of development of mass analyzers, the TOF appearsto be best-suited for the MS FC application. Ion traps (3d and linear)might be suitable provided that they are preceded by a selection device,for example a high pass mass filter, that reduces the space charge inthe trap. The array-detector magnetic sector analyzer, which offers highduty cycle and should provide high sensitivity, could be suitableprovided that an efficient array detector is developed, though at thepresent state of development the abundance sensitivity (overlap ofsignals onto neighbouring mass channels) is limiting.

The most commonly-used mass analyzer 106 coupled to the ICP is atpresent the quadrupole, principally because of its robustness, ease ofuse, and moderate cost. However, the quadrupole is a sequential scanninganalyzer having a cycle time for multiplex analysis that is longrelative to the duration of a transient signal from a single particle inthe plasma source. Therefore, the quadrupole cannot deliver correlatedmulti-analyte signals for such a short transient. A quadrupole ICP-MSanalyzer is often used for the analysis of samples presented inquasi-continuous flow, for example for nebulization and laser ablation.It is appropriate for the analysis of homogeneous samples, such as formany conventional immunoassays where total element signaling is ofinterest.

In contrast, the time-of-flight (TOF) analyzer 126 shown in FIG. 2,which samples a packet of ions in a given time period and spreads themin time according to their velocities in a potential field which are afunction of the mass-to-charge ratios of the ions, is a “simultaneous”analyzer that is suited to the analysis of short transients such asthose produced by single particles. Although TOF analyzers are known,the inventors are unaware of any TOF or other mass spectrometer analyzercurrently being used for flow cytometry. Commercial ICP-TOF-MSinstruments are some 10-100 times less sensitive than quadrupoles, atleast in part due to more significant space charge effects in the ionoptics and TOF acceleration region and to inefficiencies in duty cycle.With the employment of appropriate ion optics and other concepts notedherein, these deficiencies should be alleviated.

Another useful cytometer configuration is the OES FC 151 shown in FIG.3. A distinction between the OES FC 151 and the MS FC 101 is that in theformer, light emitted by both atoms and ions derived from the vaporizedparticle are collected and transmitted to an optical spectrometer havingan array detector. In the ICP embodiment of OES FC 151, the emission maybe collected either radially through the ICP at a specified “height”above the rf load coil (the preferred observation height is a functionof the plasma conditions, but is stable for stable ICP conditions) oraxially by looking “down” through the plasma towards the injector (whichrequires a cooled viewing interface usually with a curtain flow of gas),as shown in FIG. 3. The configuration and use of radial- andaxial-viewed ICP-OES instruments is well known by those skilled in theart.

Among the distinctions of cytometers according to the invention fromconventional fluorescence-based flow cytometry are that: (1) the cellsor beads or analytes are tagged with elements rather than fluorophores;(2) the cells or beads are vaporized, atomized and (optionally, butusually naturally under optimum conditions) ionized and it is theelemental components of the cells and beads that are detected; (3)excitation to induce emission is gained from the ICP (convective and/orelectron impact heating) rather than laser excitation at an absorptionband of the fluorophore; (4) almost all elements of the periodic tableare excited to emission (either atomic or ionic) under the operatingconditions of the ICP, whereas multiple fluorophore excitation inconventional flow cytometry generally requires two or more excitationlasers, each of which may excite one or more fluorophores withabsorption bands that are coincident with the wavelength of theexcitation laser; (5) the emitted light is dispersed by, for example,eschelle gratings or prisms in one or preferably two dimensions andcollected on an array of detectors, for example a CCD “camera”, whereasthe conventional flow cytometer uses bandpass optics to select a “leastinterfered” wavelength for each fluorophore; and (6) the emissionwavelengths are more narrow in ICP-OES than in fluorescence-based flowcytometry, and there is usually more than one usable and detectablewavelength so that inter-element interferences are both less common(better resolved emission spectra) and more easily circumvented (bychoosing an alternate emission wavelength).

EXAMPLES Example 1 Development of Aptamers for Specific LabelingLeukemic Stem Cells

Leukemic stem cells and their progenitor cells can be purified [10].They can be used as targets for selection of aptamers by selecting forthe stem cell and against the progenitor cells using a novel method ofcombinatorial screening. The selected aptamers can be tested for, andselected against cross-reactivity with other aptamers directed for themultiplex assay of the challenge. The aptamers can be labeled withdistinguishable stable isotopic elemental tags as is known to thoseskilled in the art.

Example 2 Preparation of Labeled MO7E Cell Line

A homogeneous MO7E cell-line which has been transduced with the p210bcr/abl tyrosine kinase fusion protein from chronic myeloid leukemia canbe used. This cell expresses the CD33 surface marker as well containslarge amounts of p210 internally. The markers can be tagged withantibodies or aptamers suitably tagged with commercially-availabletagging kits (NanoGold™, DELFIA™). The tagged affinity products can beincubated with fixed, permeabilized cells.

Example 3 Preparation of Quadrupole ICP-MS-Based Flow Cytometer

Demonstration of concept can be achieved using a quadrupole-based ICP-MSand the tagged cells of Example 2.

A flow cell can be constructed based on a direct injection nebulizer ora sheath-flow non-ionizing nanosprayer. A commercial flow cytometer canbe used, but with modifications, and excluding parts related tofluorescence.

Single ion monitoring at the mass/charge of one of the tag elements hasimproved duty cycle relative to scanning mode so that many of the cellevents are, observed. Subsequent measurement, in the same sample but ata later time, at the second surface tag element mass/charge will confirmindependence of the affinity chemistry and detection, with theimplication that simultaneous determination with an appropriate (TOF)detector is possible. Observation of the internal protein marker willprovide important evidence that cell volatilization is achievable. Ifthe internal marker is not detectable, in-line lysis can be used.

Example 4 Development of a Prototype Single Particle Injector

Referring to the injector 171 shown in FIG. 4, the injector is used toinject cells (or beads or other particles) 400 together with the buffersolution into the desolvation chamber 403 surrounded by a heater 405.The buffer solution flow is nebulized by high-pressure gas. The volatilecomponent of the buffer and cells (mostly water) is transferred fromaerosol to gas phase during the desolvation process and is expelled outof the desolvation chamber together with most of the nebulizer gasthrough exhaust vents 407. In most cases, the nebulizer gas flow islimited by size and design of the injector (nebulizer). Therefore, somemakeup gas can be introduced to allow complete desolvation. Desolvatedheavy cells (or beads) escape directly into the straight cylindricalchannel 409 with the rest of the gas and are introduced intovaporizer/atomizer/ionizer 104 of the EFC. In an embodiment thevaporizer/atomizer/ionizer is the ICP plasma, which allows .^(˜)1 l/minof gas to be introduced. Therefore, by adjusting the gap 411 between thedesolvation chamber and the cylindrical channel housing, one can controldesolvation as well as flow into the ICP plasma.

Example 5 Preparation of ICP-TOF-MS-Based Flow Cytometer ResearchPrototype Instrument

ICP-TOF-MS instruments are commercially available. The TOF massspectrometer provides a simultaneous analyzer which is beneficial formulitvariant analysis, of for example, rare leukemic stem cells.

An ICP-TOF-MS can be outfitted with a flow cell. The components of theinstrument as shown in FIGS. 1, 2, and 3 and described in the preferredembodiment can be assembled. Relevant components of commercial products(ELAN® ICP-MS and prOTO® orthogonal MALDI-TOF) can be procured as thebasis of a working system. Some modification of the operating systemwill be required to address the specific data collection issues of thecytometer prototype; suitable modifications are well understood by thoseof ordinary skill in the art. It can be sufficient to operateindependent computer control systems for the ICP source and the TOFanalyzer, as this would allow rapid and efficient researchinvestigation.

An instrument can be evaluated with respect to its analyticalperformance for homogeneous aqueous sample introduction as well ashomogeneous cell digests. The ICP-TOF MS-based flow cytometer can betested, for example, using human established leukemia cell lines (M07e,K562, HL-60) to investigate the capabilities with respect to the needsfor the cytometric application. Specifications for dynamic range,abundance sensitivity, transient signal pulse width and detection mode(analog/digital) for the research prototype instrument can beestablished.

The following examples have been demonstrated using a conventionalquadrupole ICP-MS instrument (sequential scanning) using conventionalnebulization of solutions obtained by acidification with HCl of thesample following immunoprecipitation and washing, which digested thesample yielding a relatively homogeneous solution. Thus, “simultaneous”determination refers in this instance to simultaneousimmunoprecipitation followed by sequential measurement of theconcomitant tags by ICP-MS.

Example 7 Dynamic Range of Anti-Flag M2 Agarose Bead Element-TaggedImmunoassay

FIG. 5 is a calibration curve of the ICP-MS linked immunoprecipitationassay of 3.times.FLAG-BAP. M2 agarose beads were used to capture samplesof serially diluted 3.times.FLAG-BAP over a concentration range of 0.05ng to 1500 ng per 100 μA 3×FLAG-BAP was detected using an anti-BAPprimary antibody and an anti-mouse-nanoAu secondary antibody. DilutedHCl was used to dissolve the nanogold tag for ICP-MS sampling. Theresults indicate that the detected signal (for gold) is linearlyproportional to the antigen (FLAG-BAP) concentration, and that at least4.5 orders of magnitude of linear dynamic range are achievable. Largedynamic range is important in the cytometric application to permitsimultaneous determination of biomarkers that appear in largelydifferent copy-counts per cell or bead.

Example 8 Simultaneous Assay of Two Cytokines Using Beads

Fluorokine™ beads coupled with cytokine capture antibodies againsteither TNF-α. or IL-6 were mixed and exposed to a mixture of cytokines,including TNF-α. and IL-6, incubated and then probed withcytokine-specific antibodies tagged with Eu (for anti-TNF-α.) and Tb(for anti-IL-6). After washing and digestion with HCl, the solution wasanalyzed for Eu and Tb. FIG. 6 provides calibration curves derived fromthis simultaneous immunoassay experiment. Linearity of signal withantigen concentration over at least 3 orders of magnitude is observed.

Example 9 Simultaneous Assay of Two Proteins Using ICP-MS-LinkedMaleylation Immunoassay

FIG. 7 shows the simultaneous quantitation of two proteins using adirect immunoassay conducted in a Reacti-bind Maleic Anhydride 96 wellplate, coupled to ICP-MS detection. In this experiment, two proteins(Human IgG and 3×FLAG-BAP) in 1×PBS were incubated in triplicate for onehour at room temperature to allow binding to the surfaces of the well ofthe Maleic Anhydride plate. Negative controls consisted of 100 μl PBSwithout protein. The plate was probed with primary antibodies anti-HumanFab′-nanoAu and anti-FLAG-Eu, washed and acidified with 10% HCl with 1ppb Ir and 1 ppb Ho as internal standards [11]. Homogeneous samples wereused wherein the elemental tag(s) are released to acidic solution forconventional nebulizer introduction to the ICP-MS. Note that thesensitivity to IgG using the nanogold tag is approximately 10 timesgreater than that for FLAG-BAP using the Eu tag; this is because eachnanogold tag contains approximately 70 gold atoms (Au is monoisotopic)while each Eu tag contained only between 6 and 10 Eu atoms,approximately equally distributed between the two natural isotopes of Eu(¹⁵¹Eu and ¹⁵³Eu, the sum of which were measured). The exampledemonstrates that at least two proteins can be immunoreactedsimultaneously and detected without mutual interference, and that thesensitivity scales with the concentration of the antigen and with thenumber of atoms of the measured isotope per tag.

Example 10 Preparation of a Kit for the Analysis of an Analyte Bound toa Single Cell by Mass Spectrometry

A kit is assembled comprising (1) a tagged biologically active materialwhich binds to an analyte of interest bound to a single cell and (2)instructions for single cell analysis by mass spectrometry.

Example 11 Forensic Applications

The methods and apparatus of the present invention can be used forforensic applications. For example, the methods and apparatus can beused to:

determine antigenic blood types (ABO and Lewis types); identify bodyfluid (blood, semen, saliva) and other biosamples (whole blood, plasma,serum, urine, cerebrospinal fluid, vitreous humor, liver or hair);

determine tissue origin (species, personal identity, etc.);

determine paternity.

Example 12 Transfusion Medicine

The methods and apparatus of the present invention can be used intransfusion medicine to:

resolve blood group A, B and D typing discrepancies; determine theorigin of the engrafted leukocytes in a stem cell recipient; and

determine the origin of lymphocytes in a patient withgraft-versus-disease.

Example 13 Flow Cytometer with ICP-MS Detector Feasibility Test

We have performed feasibility studies to validate the concept of thepresent invention. A quadrupole (sequential mass scanning) ICP-MSinstrument designed for conventional elemental analysis (and thus notoptimized for the flow cytometric application) was used. The instrumentwas modified in only two ways: a modified sample introduction system wasinstalled, and an oscilloscope was attached in parallel with signalhandling hardware and software of the original detector system.

The sample introduction system 102, 800 is shown schematically in FIG.8. Sample 400 consisting of cells or other particles was aspirated usinga syringe pump connected with capillary tubing 801 to a small volumespray chamber 803 having a drain 805 to remove condensed liquid andhaving no gas outlet except into the ICP through the 2 mm diameterinjector tube 807. Sample was pumped at 50 μL/min, about half of whichwas drained from the spray chamber 803 and half delivered to the ICP.The inventors recognize that this sample introduction device 800 may notnecessarily optimum for presentation of single particles to the ICP withhigh efficiency in all cases, depending upon the circumstances of theanalysis, but it was sufficient in this case to introduce at least afraction of the particles into the ICP and thus to show feasibility.

The discrete dynode detector of the ICP-MS instrument provides signalsthat are either analog or digital (pulse). The analog signal, taken partway along the dynode chain, is converted to digital output in thehardware and software of the detector system. The digital (pulse) signalis taken from the final dynode of the chain, is amplified and transientsignals corresponding to single pulse events whose amplitudes exceed agiven threshold are counted in the detector system hardware andsoftware. The detector system hardware and software can be configured toprovide output not of each pulse, but the integral of these over aspecified measurement period (minimum about 100 microseconds). In normaloperation, if the signal detected at the analog dynode exceeds aspecified threshold, the dynode chain downstream is disabled (disablingdigital signal detection). If the analog signal is higher than a secondthreshold, the detector firmware adjusts the voltage of the ion opticsof the instrument to defocus the ions from the detector in order toprotect the detector. An oscilloscope was tapped into the analog outputand operated in parallel with the detector hardware to enable themeasurement of the transient events over the period of a single particleevent in the ICP (e.g., up to several milliseconds with as low as a fewnanoseconds resolution).

Because the instrument used for these experiments is not capable ofmeasurement of more than one mass/charge channel during a shorttransient period, multiplex analysis of a single particle event in theICP was not be demonstrated. However, measurement of single mass/chargedetection channel events has allowed demonstration and evaluation ofcertain important characteristics of the ICP-MS detector system for thecytometric application. The inventors believe that these characteristicscan be replicated, with some differences depending on the selectedembodiment of the instrument configuration, with a simultaneous massanalyzer, with the additional benefit of facilitation of simultaneousmeasurement of many mass/charge detection channels permitting multiplexassay of single particles.

Feasibility Test 1: Detection of Single Particle Events, and Estimate ofSensitivity of Current Instrument

The MO7e cell line is a human megakaryocytic leukemia-derived cell. MO7eexpresses CD33 antigen (67 kDa single chain transmembrane glycoprotein,myeloid cell surface antigen CD33 precursor (gp67)). The cell is thoughtto express approximately 5000 to 10000 copies of antigen per cell. Thecell line was used to demonstrate that individual cells can be observedby methods according to the invention, and to estimate the sensitivityof such method using the current instrumentation. The CD33 surfacemarker was detected using monoclonal anti-CD33 (IgG1 mouse) andNanogold™-tagged anti-mouse secondary antibody (approximately 70Au-atoms per tag). It is estimated that the efficiency of secondaryantibody staining is approximately 10%.

Materials

MO7e cells were cultured for three days in a T75 flask. The cellconcentration was determined by hemocytometer and found to be 0.5.×10⁶cells/ml.

Monoclonal antibody anti-CD33, unconjugated. IgG1 (mouse) isotypesupplied at 2 mg/ml and purified in PBS/BSA with 0.1% sodium azide byImmunotech Inc. Cat #1134.

Secondary anti-mouse IgG conjugated with nanogold from Nanogold Inc.(approximately 70 Au-atoms per tag)

1% formalin prepared from 37% formalin; diluted in PBS.

Wash and antibody dilution buffer PBS/1% BSA.

50 mM ammonium bicarbonate buffer, pH 8.0.

Procedure

Tubes were soaked in PBS/1% BSA for one hour. MO7e cells were pelletedat 1500 rpm (at approximately 200 g) 5 min, resuspended in 5 ml PBS,pelleted and the wash discarded.

Cell pellet was resuspended in 3 ml PBS/1% BSA and distributed intothree eppendorf tubes (at approximately 10⁶ cells/tube) marked asprimary and secondary antibodies added; only secondary antibody added;or no antibodies added.

Primary antibody was diluted 1:50 in PBS/1% BSA and added to the cellpellet for 30 min on ice.

Cells were washed with PBS/1% BSA once.

Secondary antibody was diluted 1:50 in PBS/1% BSA and added to cellpellet for 30 min on ice.

Cells were washed once with PBS/1% BSA, once with PBS.

Live stained cells were fixed in 1% formalin/PBS for 10 min RT and leftin the fixative on ice overnight.

Cells that did not receive antibodies were treated only with PBS/1% BSAconcordantly with the stained cells.

Stained formalin fixed cells were pelleted at 1500 rpm (at approximately200 g) for 5 minutes and resuspended in 1 ml 50 mM ammonium bicarbonatebuffer, pH 8.0 per tube next day. This was discarded aftercentrifugation and fresh bicarbonate (0.5-1 ml) was added to each tube.

Tubes were vortexed gently to break up the pellet, left to sit for 5minutes for large clumps to settle to the bottom, and the top 25 μL ofwhole cell suspension were injected into the ICP-MS instrument.

Observations

The integrated (pulse detector) signal for Au for discrete cellintroduction gave 300-500 counts per second (cps), secondary antibodiesonly, less than 100 cps, no antibodies, less than 10 cps and bufferonly, less than 3 cps.

FIG. 9 shows the overlaid results of separate direct injections of the100 ppt Rh (1% HNO₃) and cell suspension (separate injection, 50 mMNH₄HCO₃) samples as described above.

FIGS. 10A and 10B show oscilloscope data associated with FIG. 9. FIG.10A shows the signal for Ar₂+(about 10⁷ cps) signal. The upper tracecovers a relatively large time window (ca. 100 .μs), from which onecould conceivably determine the average ion signal rate. The lower traceshows the pulse for a single ion detection event (over a greatlymagnified time scale). FIG. 10B shows Au⁺ from cell introduction. Theupper trace indicates that multiple ion signal pulses are not observed.The lower trace shows the signal pulse for a single Au ion detectionevent. Typically, only one ion pulse was observed in a particle eventtime window, suggesting that we detect on average only about one Au atomper cell.

Efficiency of Detection

We estimate the sample introduction rate at (very) approximately 400cells/second, derived as follows: approximately 1×10⁶ cells per sample,1 mL/sample at 50 .μL/minute introduction sampling with 25 .μL/mindelivered to the ICP.

Accordingly, we infer that approximately one Au atom per cell isobserved.

The detection efficiency of the instrument used was estimated from thesignal obtained from continuous aspiration of a sample containing 100parts per trillion (ppt, mass/volume) in 1% nitric acid. The signalobtained was approximately 2000 cps, suggesting an efficiency fordetection of Rh of 1×10⁻⁵, derived as: 100 ppt (10⁻¹⁰ g/mL), atomicweight 103 g/mole, 25 μL/min delivered to plasma, yielding 2×10⁸ atomsRh/second delivered to plasma for which 2000 cps is observed. Theinventors ascribe this efficiency to the following: 100% ionization, 1%transmission through the vacuum interface (100% of central plasmacontaining ion inhaled through sampler, 1% of sampler flow transmittedthrough skimmer), 10% transmission through mass analyzing quadrupole,and therefore about 1% transmission through the ion optics. (From theseestimates, we infer that improvements in sensitivity for the cytometricapplication, assuming retention of the vacuum interface configuration,should principally focus on improving the transmission through the massanalyzer (e.g., TOF with high duty cycle) and, more importantly, the ionoptics (according to the earlier discussion, principally throughaccelerating optics and elimination of space-charge-inducing ions)).

Therefore, if one Au atom detection event per cell is obtained, and thisis obtained with the same detection efficiency as Rh solution, weestimate that the MO7e cell averages approximately 1400 tagged CD33markers per cell. With the assumption that the efficiency of the twoantibody tagging is about 10%, the estimated number of CD33 per cell is14000, which is consistent with the 5000-10000 quoted earlier. It isdesirable to provide higher sensitivity so that proteins of lowercopy-count per cell can be detected. In addition to the ion opticalimprovements suggested above, direct immuno-tagging (as opposed to the 2antibody sandwich used here) is expected to be advantageous.

We conclude that the method is able to detect single particle events inthe plasma. The experiments described provide guidance for researchefforts to improve the sensitivity of the method. A simultaneous massanalyzer is required to facilitate the multiplex advantage that the massspectrometer detector provides to flow cytometry.

Feasibility Test 2: Estimation of the Transient Period of a SingleParticle Event

The MO7e cell sample used in Feasibility Test 1 provides an opportunityfor the estimation of the transient period of a single particle event,which is important for the design and optimization of the MS FC. It isestimated that the NaCl content in the cell is 0.9% w/w. For a 16micrometer cell this converts to 2×10¹¹ atoms of Na per cell. Theefficiency of the instrument used in these experiments for Na detectionis lower than for Rh; about 1×10⁻⁶. Thus, for a single cell event, 2×10⁵ions will reach the detector. This is a sufficiently-large number thatthe arrival period of Na ions corresponding to a single MO7e cell eventcan be measured.

If the transient produced by the single cell event is of the order of100-300 microseconds (as reported by Olesik for monodispersed 3-65micrometer particles), an equivalent average count rate of (0.7-2)×10⁹is achieved (with peak current about twice that).

FIG. 11A shows the Na⁺ signal detected at the oscilloscope over theperiod of several cell introduction events. The data given in FIG. 11Ashows the Na⁺ signal when cells are introduced in a 30 mM CaCl₂ buffer.The data shown in FIG. 11B presents the results for buffer only. Thevariability of the observed signals may reflect the variability in cellsize (volume and thus Na content) of the cell population, or mightindicate the presence of Na-containing particles other than MO7e cells.The important observation, for the present purposes, however, is thatthe transient signal for a single particle event is of the order of100-150 μs.

The baseline is very different between the two datasets shown in FIGS.11A and 11B. This difference can be attributed to the fact that a firstcell detected should trip the higher threshold detector protectioncircuitry and activate ion defocusing. This is because the anticipated2×10⁵ Na ions per cell arrive in the period of approximately 100-150 μs,which corresponds to an average count rate exceeding 10⁹ per second, issufficient to trip the second threshold detector. In the absence ofcells in the sample (data given on the right in FIG. 11), the detectorprotection circuitry is not tripped. The ion optical defocusing appearsto suppress ion transport by about a factor of 1000, accounting for thedifference in the baseline data, but this is not intended to be a stableor reproducible (quantitative) defocusing factor.

Transient signals of 100-150 μs period, ascribed to MO7e introductionevents, were observed at a frequency of about 5 to 6 per 10milliseconds, or about 500 to 600 cells per second. This is consistentwith the estimates made earlier, and with the estimate of 10⁶ cells per1 mL in the original sample (procedure step 4 of Feasibility Test 1,subsequently reduced to approximately 1 mL volume in step 9).

A notable inference taken from this experiment is that the high Na⁺signal anticipated for cells, or effects related to the change in massdistribution of the plasma ions as a result of the passage of a particlethrough the plasma, might provide a means to trigger the system upon acell event. Further, it is feasible that the magnitude of the Na⁺ signal(or signal of another element at high concentration in the cell), or themagnitude of effects related to the ion distribution change as a resultof the particle's passage through the plasma, could be correlated withthe physical size of the particle, which may be of importance inidentifying target particles or distinguishing single particles fromgroups of particles.

The important conclusion of this experiment is that the transient signalis approximately 100-150 μs FWHM in duration. This has implications fordesign considerations to provide dynamic range. Further, it is evidentthat particles characterized by transient signals of this period can beintroduced to the system at a rate of about 3000 per second (so thatsignal corresponding to a particle is present up to 50% of the time).Smaller cells, and smaller beads, should have shorter transients, andthus allow higher rate of introduction.

Feasibility Test 3: Comparison of Current FACS with the Current ICP-MSWith Cell Injection, and Demonstration of Entire Cell Volatilization

The inventors have had an opportunity to compare directly theperformance of a current FACS instrument to that of the ICP-MSinstrument with cell injection described in Feasibility Test 1. Further,the test was configured to provide for tagging of intracellularproteins; if these internal tags can be detected, this implies that theentire cell and its contents were vaporized, atomized and ionized,rather than just vaporization of surface tags.

Because the ICP-MS instrument used for these experiments was not asimultaneous detector, the same (nanogold) tags could be used for eachantigen, and immuno-tagging was performed in separate vials for eachsample and antigen. Thus, each antigen for each sample was determined ina separate analysis. Samples were introduced to the ICP-MS as describedin Feasibility Test 1.

Preparation of Samples for ICP-MS Analysis with Cell Injection

Materials

Human Monocyte Cell Lines:

MO7e parent line is a human megakaryocytic leukemia-derived cell. MO7eexpress CD33 antigen (67 kDa single chain transmembrane glycoprotein,myeloid cell surface antigen CD33 precursor (gp67)). Approximately5000-10000 copies of CD33 antigen per cell.

MBA-1 and MBA-4 are stable clones of MO7e transfected with p210 BCR/Ablexpression plasmid.

HL-60 (ATCC cat #CCL-240), myeloid leukemia cell line used as antigen inproduction of anti-CD33 monoclonal antibodies.

Antibodies:

anti-CD33, mouse monoclonal, unconjugated. IgG1 (mouse) isotype.Supplied at 2 mg/ml purified in PBS/BSA with 0.1% sodium azide(Immunotech Inc. Cat #1134)

anti-IgG2a, mouse, (BD PharMingen, cat #555571) (0.5 mg/ml stock)

anti-BCR antibody raised in rabbit (Cell Signaling Tech. Cat#3902), usedat 1:25 for flow cytometry

Secondary antibodies: 2001 nanogold-anti-mouse IgG (NMI) and 2004nanogold anti-rabbit Fab′ (NRF) (Nanoprobes Inc.) used at (1:50)according to manufacturer's recommendation.

Buffers:

BD Biosciences FACS permeabilization solution 2 (cat #347692) PBS withCa++/Mg++;

PBS/1% BSA

1% and 0.5% formalin prepared from 37% formalin; diluted in PBS 50 mMammonium bicarbonate buffer, pH 8.0

Procedure:

Tubes were soaked in PBS/1% BSA for one hour.

Cells were pelleted at 1500 rpm (^(˜)200 g) 5 min, resuspended in 5 mlPBS and counted using a hemocytometer. Cell yield:

M07e-1e6/ml

MBA-1-1e6/ml

MBA-4-1e6/ml

HL-60-1e6/ml

M07e (tube #1) and HL-60 (tube #2) were stained live with anti-CD33(1:50) on ice for 30 min; followed by one wash with PBS/BSA.Anti-mouse-IgG-Au (1:50) was added to the washed cell pellet for another30 min on ice. Live stained cells were fixed in 1% formalin/PBS for 10min RT and left in the fixative on ice over 48 hours.

MBA-1 (tube #3), MBA-4 (tube #4) and MO7e (tube #5) were permeabilisedand fixed in the FACS Permeabilization Solution 2 for 10 min at RT.

After one wash the cells were incubated in media with 10% FBS to blocknon-specific antigen sites for 15 min RT.

Permeabilized cells were treated with anti-BCR antibodies (1:25) (tubes#3,4,5) or with non-specific IgG (tubes #3°, 4°, 5°. w/o primaryantibody) for 45 min RT. Secondary antibodies were added to washed cellsanti-rabbit-IgG-Au (1:50) for 45 min RT.

Stained cells were washed twice prior to post-fixation in 0.5% formalinand kept in fridge over the weekend prior to MS analysis when theformalin was replaced with 50 mM ammonium bicarbonate.

Preparation of Samples for FACS Analysis (Carried Out Simultaneouslywith Above)

Materials

Antibodies:

anti-IgG1-FITC mouse isotype, (BD PharMingen)

anti-CD45-FITC antibody raised in mouse (BD PharMingen) used at 1:50 forflow cytometry. CD45 is expressed on the surface of all humanleukocytes. Used as a positive sample for FACS set-up.

Secondary fluorescent antibodies: anti-mouse IgG-FITC (BD PharMingen)and anti-rabbit-FITC (Biolab) used at (1:50)

Buffers:

BD Biosciences FACS permeabilization solution 2 (cat #347692)

PBS with Ca++/Mg++;

PBS/1% BSA

1% and 0.5% formalin prepared from 37% formalin; diluted in PBS 50 mMammonium bicarbonate buffer, pH 8.0

Procedure

Cell preparation and primary antibody staining was done in parallel withsamples for ICP-MS with 1e6 cells/ml/tube.

All procedures with fluorescent secondary antibody staining and cellwashes were carried out in the dark on ice.

After the final PBS wash cells were resuspended in PBS (not formalin)and immediately processed by FACS (BD FACSCalibur).

Gates and settings were determined using the anti-CD45-FITC stainedHL-60 as positive (R4) channel and isotype anti-mouse IgG-FITC stainedHL-60 as negative (R3) channel.

Observations

The results for both the ICP-MS detection (shaded grey) and conventionalFACS (white) are summarized in FIG. 12.

Standard deviations for triplicate analyses by ICP-MS are shown by errorbars; equivalent uncertainties for the FACS results were not provided.

Both the CD33 (surface markers on MO7e and HL60) and the BCR (internalmarker in MO7e, MBA1 and MBA4) were determined by both FACS and ICP-MSdetectors. This implies that the entire (permeabilized) cells and theircontents were vaporized, atomized and ionized in the ICP-MS. Further,the FACS and ICP-MS results are largely in rather good agreement forboth the surface and internal markers and for the procedural blanks

We conclude from these results that FACS and ICP-MS detection (using thecurrent un-optimized instrument) provide comparable results for singleantigen assay. It is anticipated that the sensitivity of the ICP-MSdetector will be improved as discussed above, and that incorporation ofa simultaneous mass analyzer will permit high order multiplex assay. Itis also evident that the MO7e, MBA1 and MBA4 cells used in theseexperiments were efficiently vaporized, atomized and ionized. Thissuggests that the optional in-line lysis device discussed above is notrequired for these or similar cells.

Feasibility Test 4: Production and Detection of Element-Tagged Beads

Another approach to multiplexed assay is to use different identifiablebeads that immobilize antigens. The beads typically have captureaffinity agents (e.g., antibodies) attached to their surface. Afterexposure to a sample, the bead-antigen complexes are typically exposedto a second affinity product (antibody, aptamer, etc.) which is taggedwith an element or isotope as already discussed (herein, and in U.S.patent application Ser. No. 09/905,907, published under US 2002/0086441on Jul. 4, 2002 and Ser. No. 10/614,115). The beads are distinguished bytheir elemental composition, which might be a surface element label, andencapsulated element label or an element label incorporated within thebead material. The identity of the bead can be associated with the typeof capture affinity agent attached to the bead or to the sample (e.g.,beads with different element labels are exposed to different samples, orare placed in different wells of a 96- or 384- or 1536-well plate).Thus, detection of the secondary affinity product tag determines thepresence of the antigen and the element composition (element label) ofthe bead indicates which antigen was captured or the sample in which itwas captured. The method is modeled after U.S. Pat. No. 6,524,793,assigned to Luminex, and references therein.

The beads may be of any appropriate material (e.g., polystyrene,agarose, silica). Each bead may contain one or more affinity captureagents, and multiplexed assay of the antigens captured on the bead maybe conducted. The element label incorporated in or on the bead may be asingle element or isotope or, preferably, a combination of elements orisotopes. For example, if the dynamic range of the detector is threeorders of magnitude and differences in signal levels of a factor ofthree are reliably detected, two element labels can be combined indifferent ratios to provide 63 distinguishable beads. Under the sameconditions, 5 element labels can provide 32,767 distinguishable beads.With 5 orders of dynamic range and 5 element labels for which factors ofthree in signal can be reliably detected, 248,831 distinguishable beadscan be constructed. It will be recognized that the beads can bemanufactured to a size suitable for complete vaporization, atomizationand ionization in the device used for that purpose (e.g., ICP). It willalso be recognized that smaller beads are likely to provide shortertransient signals, and that accordingly the rate of particleintroduction can be optimized for the particular beads used.

To demonstrate the viability of the method, stober silica particleshaving a diameter of about 150 nm were grown in various lanthanide (Ho,Tb, Tm) solutions. The lanthanide elements were incorporated into thesilica particles. The silica particles (beads) were introduced seriallyto the ICP-MS instrument as described in Feasibility Test 1. Since theinstrument used was not capable of simultaneous multielement analysis,the transient signals for the lanthanides and for silicon were measuredseparately for different beads.

FIGS. 13A and 13B show some of the data obtained. The data provided inthe FIG. 13A shows the detection of Si⁺, clearly indicating that thebeads are vaporized, atomized and ionized. Data provided in FIG. 13Bshow the detection of Tb⁺ (for beads grown in Tb solution). Clearly, theTb label is detected. If a mixture of beads having different lanthanidelabels were sampled, the different lanthanide signals would identify thedifferent beads. It is also evident that beads can be grown in solutionsof mixed lanthanides (or other elements), and would incorporate thedifferent elements, thus providing for a larger number ofdistinguishable beads as indicated above. The availability of asimultaneous analyzer would further allow simultaneous detection of theelements associated with the bead itself and also with the tagassociated with a secondary affinity product that recognizes a capturedantigen.

Therefore, elements within a bead can be detected (i.e., the bead isvaporized to its atomic components). Different combinations of elementinternal “labels” can be used to distinguish beads. If those beads carrydifferent surface antibodies to bind different antigens, and thoseantigens are then recognized by another antibody containing a differentelement reporter tag, a multiplexed assay is enabled. Alternatively, thedifferently labeled beads can be used with the same surface antibodies,but with the different beads being applied to different samples (such asa 96 well plate), so that the signal associated with the labeledaffinity product identifies the antigen concentration in the sampleindicated by the signals corresponding to the bead composition. Numerousmodifications, variations and adaptations may be made to the particularembodiments of the invention described above without departing from thescope of the invention, which is defined in the claims.

REFERENCES

The following publications are hereby incorporated by reference

-   1. Hanayama, R.; Tanaka, M.; Miwa, K.; Shinohara, A.; Iwamatsu, A.;    Nagata, S. Identification of a factor that links apoptotic cells to    phagocytes. Nature 2002, 417, 182-187.-   2. Reif, K.; Ekland, E. H.; Ohl, L.; Nakano, H.; Lipp, M.; Forster,    R.; Cyster, J. G. Balanced responsiveness to chemoattractants from    adjacent zones determines B-cell position. Nature 2002, 416, 94-99.-   3. Heppner, F. L.; Musahl, C.; Arrighi, I.; Klein, M. A.; Rulicke,    T.; Oesch, B.; Zinkemagel, R. M.; Kalinke, U.; Aguzzi, A. Prevention    of scrapie pathogenesis by transgenic expression of anti-prion    protein antibodies. Science 2001, 294, 178-182.-   4. Shinkai, K.; Mohrs, M.; Locksley, R. M. Helper T cells regulate    type-2 innate immunity in vivo. Nature 2002, 420, 825-829.-   5. Marx, J. Mutant stem cells may seed cancer. Science 2003 301:    1308-1310.-   6. Lapidot, T. C. Sirard J. Vormoor, B. Mudoch T. Hoang, J.    Caceres-Cortes, M. Minden, B. paterson, M. Caligiuri, and J. E.    Dick. A cell initiating human acute myeloid leukaemia after    transplantation inot SCID mice. Nature 1994 367, 6464: 645-648.-   7. Meldrum, D. R.; Holl, M. R. Tech. Sight. Microfluidics.    Microscale bioanalytical systems. Science 2002, 297, 1197-1198.-   8. Schenk, T.; Molendijk, A.; Irth, H.; Tjaden, U. R.; van    der, G. J. Liquid chromatography coupled on-line to flow cytometry    for postcolumn homogeneous biochemical detection. Anal. Chem. 2003,    75, 4272-4278.-   9. S. D. Tanner. Space charge in ICP-MS: Calculation and    implications. Spectromchimica Acta 1992 47B: 809-823.

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What is claimed is:
 1. An elemental flow cytometer, comprising: a deviceto vaporize, atomize, and ionize material; an introduction system forintroducing packets of discrete entities into said device to vaporize,atomize and ionize materials to vaporize, atomize and ionize theentities in the packets; and a spectrometer adapted to individuallyanalyze elemental composition of one or more of the vaporized, atomizedand excited or ionized packets.
 2. An elemental flow cytometer as setforth in claim 1 wherein the introduction system comprises a laserablation system.
 3. An elemental flow cytometer as set forth in claim 2wherein the laser ablation system is adapted to produce said packets bydirecting laser pulses toward samples and thereby producing discreteentities of ablated material to form the packets.
 4. An elemental flowcytometer as set forth in claim 1 wherein the introduction systemcomprises an in-line lysis system.
 5. An elemental flow cytometer as setforth in claim 4 wherein the in-line lysis system is adapted to producesaid packets of discrete entities by rupturing cells or other particles.6. An elemental flow cytometer as set forth in claim 1 wherein thedevice to vaporize, atomize, and ionize comprises an inductively coupledplasma.
 7. An elemental flow cytometer as set forth in claim 1 whereinthe device to vaporize, atomize, and ionize is adapted for operation atatmospheric or higher pressure.
 8. An elemental flow cytometer as setforth in claim 1 wherein the device to vaporize, atomize, and ionize isadapted to substantially completely ionize the entities in the packets.9. An elemental flow cytometer as set forth in claim 1 wherein theintroduction system is adapted to introduce the packets sequentiallyinto the device to vaporize, atomize, and ionize the entities in thepackets.
 10. An elemental flow cytometer as set forth in claim 9 whereinthe introduction system is adapted to introduce the packetssubstantially one at a time into the device to vaporize, atomize, andionize.
 11. An elemental flow cytometer as set forth in claim 1 whereinthe packets contain a predetermined number of discrete entities.
 12. Anelemental flow cytometer as set forth in claim 1 wherein thespectrometer comprises a mass spectrometer.
 13. An elemental flowcytometer as set forth in claim 12 wherein the mass spectrometercomprises a time-of-flight mass spectrometer.
 14. An elemental flowcytometer as set forth in claim 13 wherein the time-of-flight massspectrometer is configured for orthogonal extraction of ions from an ionbeam produced by the device to vaporize, atomize, and ionize.
 15. Anelemental flow cytometer as set forth in claim 12 wherein the massspectrometer comprises a magnetic sector mass spectrometer.
 16. Anelemental flow cytometer as set forth in claim 12 wherein the massspectrometer is adapted to detect transient signals produced by analysisof distinct ion clouds associated with the ionization of individualpackets and to maintain data relating to an individual packet distinctfrom data relating to other packets to enable analysis of the contentsof the individual packets.
 17. An elemental flow cytometer as set forthin claim 12 further comprising an ion pre-treatment system adapted totransport ions from the device to vaporize, atomize, and ionize to themass analyzer.
 18. An elemental flow cytometer as set forth in claim 17wherein the ion pretreatment system comprises a high pass ion guideadapted to limit transportation of plasma support gas ions to the massanalyzer.
 19. An elemental flow cytometer as set forth in claim 18wherein the ion guide is adapted to allow passage of ionized Lanthanideions.
 20. An elemental flow cytometer as set forth in claim 12 furthercomprising a trigger system adapted to trigger a detection system of themass analyzer to acquire data associated with a discrete packet inresponse to detection by the trigger system of an event associated withan ion cloud produced as the discrete entities in said packet areionized by the device to vaporize, atomize, and ionize.